S Polat and others Three novel CYP11B1 mutations 170:5 697706

Clinical Study

Characterisation of three novel CYP11B1

mutations in classic and non-classic
11b-hydroxylase deficiency
Seher Polat, Alexandra Kulle1, Zuleyha Karaca2, Ilker Akkurt3, Selim Kurtoglu4,
Fahrettin Kelestimur2, Joachim Grotzinger5, Paul-Martin Holterhus1 and
Felix G Riepe1
Department of Medical Genetics, Erciyes University, Kayseri, Turkey, 1Division of Pediatric Endocrinology and Correspondence
Diabetes, Department of Pediatrics, University Hospital Schleswig-Holstein, Christian-Albrechts-University Kiel, should be addressed
Schwanenweg 20, D-24105 Kiel, Germany, 2Department of Endocrinology, Erciyes University, Kayseri, Turkey, to F G Riepe
3
Childrens Hospital Altona, Pediatric Endocrinology, Hamburg, Germany, 4Department of Pediatric Endocrinology, Email
Erciyes University, Kayseri, Turkey and 5Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany friepe@pediatrics.uni-kiel.de

Abstract
European Journal of Endocrinology

Background: Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited
endocrine diseases. Steroid 11b-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH.
Aim: The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations
(p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate
this data with the clinical phenotype.
Methods: Functional analyses were done by using a HEK293 cell in vitro expression system comparing WT with mutant
P450c11 activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of
the protein.
Results: Two mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a
complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed
partial functional impairment with 19% of WT activity.
Conclusion: Functional mutation analysis enables the correlation of novel CYP11B1 mutations to the classic and non-classic
11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to
reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing
number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis
in patients with otherwise unexplained hyperandrogenism.

European Journal of
Endocrinology
(2014) 170, 697706

Introduction
Congenital adrenal hyperplasia (CAH), one of the
most common autosomal recessive inherited endo-
crine diseases, is characterised by complete or partial
impairment of adrenal steroidogenesis (1, 2). Although
over 90% of cases of CAH are caused by 21-hydroxylase
deficiency, steroid 11b-hydroxylase (P450c11, EC
1.14.15.4) deficiency (11OHD) accounts for 58% of
cases, reflecting a frequency of w1:100 0001:200 000
live births in non-consanguineous populations (3, 4, 5).
The 11b-hydroxylase belongs to the cytochrome
P450 system (P450c11) that facilitates the conversion of
11-deoxycortisol (S) to cortisol (F) and 11-deoxycortico-
sterone (DOC) to corticosterone (B) in the mitochondria
of the adrenal cortex. 11OHD is characterised by deficient

www.eje-online.org 2014 European Society of Endocrinology Published by Bioscientifica Ltd.

DOI: 10.1530/EJE-13-0737 Printed in Great Britain
 Clinical Study S Polat and others Three novel CYP11B1 mutations 170:5 698

cortisol synthesis caused by mutations in the CYP11B1
gene coding for 11b-hydroxylase (6, 7, 8, 9). An impair-
ment of 11b-hydroxylase activity leads to an accumulation
of the precursor steroids, which are shunted into the
adrenal androgen synthesis pathway, resulting in prenatal
virilisation of female external genitalia (46,XX disorder
of sex development). Moreover, the excess of postnatal
androgen causes androgenisation and rapid somatic
growth as well as accelerated bone maturation in both
sexes. The accumulation of DOC, which binds to and
activates the mineralocorticoid receptor, leads to hyper-
tension in about two-thirds of all patients (3, 10, 11, 12,
13). Hormonal diagnosis of 11OHD is verified by elevated
baseline concentrations of DOC and S in classical forms
as well as increased response to cosyntropin stimulation
in non-classic forms (14).
The non-classic 11OHD form is caused by partial
impairment of the P450c11 function (10, 15) with a
phenotype resembling non-classic 21-hydroxylase defici-
European Journal of Endocrinology
11OHD (10). Unlike in classic 11OHD, arterial hyperten-
sion is not commonly found.
In the current study, three novel CYP11B1 mutations
detected in three patients with classic and in one patient
with non-classic 11OHD were studied in order to describe
the effect of these variants.
Subjects and methods
Patient 1
A male patient (karyotype 46,XY) of Turkish origin was
referred to the Paediatric Endocrinology Department at
Erciyes University, Kayseri, Turkey at the age of 2 years with
the complaint of penile growth. His parents are first cousins
and he has three siblings, one of them also suffering from
11OHD (patient 2). Results of the initial physical exami-
nation were as follows: height 97 cm (C3.1 SDS) (16),
weight 17 kg, BMI 18 kg/m2 (C1.07 SDS) (17), blood

ency (11). Non-classic 11OHD can manifest with mild
virilisation and precocious pseudopuberty in children.
Female patients with non-classic 11OHD are born with
normal external genitalia and may have hirsutism and
oligomenorrhea in adulthood, resembling signs and
symptoms suggestive for polycystic ovary syndrome.
However, only a minor percentage of women with
hirsutism and oligomenorrhea suffer from non-classic
pressure 100/70 mmHg (90th percentile) (18), testicular
volume 3 ml, penile length 9 cm (C5.3 SDS) (19), advanced
bone age of 7 years and dark skin pigmentation especially at
the genitalia. Abdominal ultrasound examination revealed
hyperplasic adrenal glands. Steroid hormone analyses
supported the diagnosis of 11OHD (Table 1). Hydrocorti-
sone supplementation was initiated. During his last visit
at 14 years of age, physical examination revealed the

Table 1 Laboratory data before treatment.

Patient 1 Patient 2 Patient 3 Patient 4

Karyotype 46,XY 46,XX 46,XY 46,XX

Genotype p.His125Thrfs*8/ p.His125Thrfs*8/ p.Leu463_Leu464dup/ p.His125Thrfs*8/
p.His125Thrfs*8 p.His125Thrfs*8 p.Leu463_Leu464dup p.Ser150Leu
Age at analysis 2 years 2 days 26 years 5 years
11-Deoxycorticosterone ND ND ND 1.0/11.5 (bl. 0.060.52)
(ng/ml)
Corticosterone (ng/ml) ND ND ND 1.4/22.5 (bl. 0.091.46)
Aldosterone (pg/ml) ND 258 (70540) 16.7 (35300) ND
17-OHP (ng/ml) 17.56 (0.63.4) 48.73 (0.63.4) 4.2 (0.63.4) 0.40/1.9 (bl. 0.060.56)
11-Deoxycortisol (ng/ml) 60.35 (0.114) 31.94 (0.114) 46.6 (08) 4.92/30.9 (bl. 0.091.11)
Cortisol (mg/dl) 38a (526) 73.7a (526) 2.2 (526) 90/158 (bl. 5.62170.28
and stim. O150)
DHEA-S (ng/ml) 532 (50480) 4692 (50480) 7527 (3003330) ND
Androstenedione 23.66 (0.080.5) 70.10 (0.22.9) ND 109/97 (bl. 2.943)
(ng/ml)
Total testosterone 657 (310) 4355 (2064) 492 (1180) 26/34 (bl. 2.920)
(ng/dl)
Renin (pg/ml) ND 0.76 (1.96.0) 0.89 (1.96.0) ND
ACTH (pg/ml) 2690 (560) 1540 (560) 4328 (560) ND

Reference ranges in brackets (bl., baseline and stim., stimulated), ND, not done. Steroids in the patients 1, 2 and 3 have been detected by RIA.
a
The cortisol RIA (ICN Biomedicals, Inc., Irvine, CA, USA, 1999) assay used in patients 1 and 2 has a 33% cross-reactivity with 11-deoxycortisol
causing elevated cortisol concentrations in the case of 11OHD. Steroids in the patient 4 have been detected by LCMSMS (56) before and 60 min after 250 mg
ACTH i.v.

www.eje-online.org
 Clinical Study S Polat and others
following: height 169 cm (0.1 SDS) (20), weight 59 kg, BMI
20 kg/m2 (C0.37 SDS) (17), blood pressure 110/70 mmHg
(50th percentile) (17), bilateral testicular volume 25 ml,
pubic hair Tanner stage 5, stretched penile length 13 cm
(C0.0 SDS) (18) and a bone age of 16 years. Although he
was advised to take up 20 mg/m2 per day hydrocortisone
by the general practitioner, the laboratory investigations
revealed insufficient control of the disease due to non-
adherence to the therapy (DOC 215.81 ng/ml (reference
range (0.121.58), DHEAS 981 ng/ml (12003700), total
testosterone 882 ng/dl (350970) and androstenedione
9.42 ng/ml (0.331.9)).
Patient 2
The female patient (karyotype 46,XX) was born after
an uneventful pregnancy (height at birth 50 cm, weight
3400 g). She is the younger sister of patient 1. She was
referred at the age of 2 days to the Paediatric Endocrinology
Department at Erciyes University, Kayseri, Turkey because
European Journal of Endocrinology
of ambiguous genitalia. She had virilised external genitalia
reflecting Prader stage 3 without obvious hyperpig-
mentation of the skin. Pelvic ultrasonography revealed a
small uterus, cervix and vagina. Steroid hormone analyses
confirmed the diagnosis of 11OHD (Table 1). Hydro-
cortisone treatment was initiated. At her last visit to the
Paediatric Endocrinology Department at the age of
12.5 years, the physical examination results were weight
42 kg (K0.1 SDS), height 145 cm (K1.04 SDS) (20), BMI
20 kg/m2 (C0.17 SDS) (21), blood pressure 90/50 mmHg
(10th percentile) (18), pubic hair and breast develop-
ment Tanner stage 3 and bone age of 14 years. The patient
was only irregularly followed up and adhered poorly to
the treatment with hydrocortisone, which was increased
up to 20 mg/m2 per day by the general practitioner.
Hormone analyses showed insufficient therapy (DOC
281 ng/ml (reference range 0.121.58), DHEAS 482 ng/ml
(3202260), total testosterone 269 ng/dl (1535) and
androstenedione 7 ng/ml (0.51.7)).
Patient 3
A 27-year-old male patient (karyotype 46,XY) of Turkish
ancestry was referred to the Endocrinology Department
at Erciyes University, Kayseri, Turkey with the complaint
of infertility (Table 1). Premature pubarche led to clinical
work up in infancy and revealed 11OHD. Hormonal
data from that time are not available. Treatment with
hydrocortisone was initiated at that time, but then he was
lost to follow-up until the age of 27 years. At that time,
Three novel CYP11B1 mutations 170:5 699
his height and weight were 152 cm (K3.41 SDS) (20) and
65 kg, respectively, and his BMI was 28 kg/m2 (C0.11 SDS)
(17). Hormonal data at 27 years of age is shown in Table 1.
Blood pressure was not taken on that occasion. Computed
tomography scan of adrenal glands revealed nodular
thickening. Semen analysis revealed severe oligospermia
and mild-to-moderate asthenozoospermia. Follicle-
stimulating hormone and luteinising hormone levels
were elevated with 17 mIU/ml (reference range 1.512.4)
and 10 mIU/ml (1.78.6) respectively. Scrotal ultrasono-
graphy revealed two heterogeneous hypoechogenic
lesions (right testis, 38!20 mm and left testis, 24!
155 mm) with lobulated contours and irregular
boundaries reflecting testicular adrenal rest tumours
(TART). The TART located on right testis was removed
by testis sparing surgery. Histological examination was
compatible with TART (Fig. 1). Prednisolone therapy was
resumed and the patients wife gave birth to a female
child. The patients fertility was not investigated in detail
as he refused to perform a semen analysis.
Patient 4
The female patient (karyotype 46,XX) of Moroccan
descent was diagnosed at the Paediatric Endocrinology
unit at the Childrens Hospital Altona, Hamburg,
Germany, as non-classical 11OHD when she was
5.5 years old and came to attention with premature
Figure 1
Histopathology from frozen tissue section of TART
(magnification !40). Multiple nodules are present having
an appearance comparable with adrenal origin. The lesion
was composed of sheets and nests separated by fibrous tissue.
The individual cells were large, round with round nuclei with
defined borders and abundant eosinophilic cytoplasm
(cutout, magnification !100).
www.eje-online.org
 Clinical Study S Polat and others
pubarche without further signs of virilisation. Clinical
characteristics at diagnosis were height 112, 2 cm (K0.7
SDS) (20), growth rate 10.7 cm/year (C5.2 SDS), weight
21.3 kg, BMI 16.9 kg/m2 (0.86 SDS) (17) and bone age of
8.5 years. Ultrasonography of the ovaries and adrenals
revealed no pathological changes. Baseline DOC and S
were elevated and cortisol was within the reference range
(Table 1). ACTH stimulation test showed significantly
increased response of DOC and S with sufficient rise of
cortisol compatible to non-classic 11OHD. Treatment
with hydrocortisone (10 mg/m2 per day) was initiated.
At the age of 7.1 years, breast development started and
therapy with triptorelin was started without performing
further hormonal tests because of precocious puberty
secondary to adrenal androgen excess.
The brother of patient 4 was first seen at the age of
8 months. Auxological data were height 68 cm (K1.1 SDS)
(20), weight 9 kg and BMI 19.5 kg/m2 (C1.1 SDS) (17). The
androgen metabolites in urine were within the normal
range for age. At the age of 16 months there were still
European Journal of Endocrinology
no signs of virilisation, height 82.2 cm (C0.4 SDS) and
growth rate accelerated with 20.3 cm/year within the
last 8 months (C1.5 SDS). Bone age was not analysed yet.
Sequence analysis of the CYP11B1 gene
The molecular analysis of the CYP11B1 gene was carried
out after receiving informed consent from patients and/or
legal guardian for genetic studies. Genomic DNA was
prepared from peripheral blood leukocytes and amplifi-
cation of exons 19 of the CYP11B1 gene was carried out
as described previously (22, 23). Direct DNA sequencing
and analysis of the coding region of the CYP11B1 gene
was done as described previously (23). Sequence variants
were designated according to the recommendations of
the Human Genome Variation Society (www.hgvs.org/
rec.html) using the GenBank reference sequences
NC_000008.10 (CYP11B1 g.DNA), NM_000497 (CYP11B1
c.DNA) and NP_000488.3 (CYP11B1 p.protein).
Transient transfection assay
Each mutation was introduced into a CYP11B1-pcDNA3.1
expression vector construct (kindly provided by Prof. R
Bernhardt, Institute of Biochemistry, University des
cken, Germany) by site-directed muta-
Saarlandes, Saarbru
genesis as described previously (24, 25). In vitro transient
transfection assay was performed in triplicates using
the HEK293 cell line thrice. The cells were transfected
with each pcDNA3.1-CYP11B1 mutant constructs with
www.eje-online.org
Three novel CYP11B1 mutations 170:5 700
additional transfection of adrenodoxin (pECE-ADX), Adx
reductase (pECE-ADR) expression vectors (kindly provided
by Prof. W L Miller, Department of Pediatrics, University
of California, San Francisco, CA, USA) and pRK-TK
(Promega) coding for Renilla luciferase as it was described
in our previous paper (25). The kinetic constants of
CYP11B1 in intact HEK293 cells were determined 48 h
after transfection. The cells were incubated in triplicates
for 270 min at 37 8C with 1 ml DMEM medium containing
0.1, 0.25, 0.5, 1, 2, and 4 mmol/l DOC and S in parallel
with 10 mM NADPH (SigmaAldrich). The steroid hor-
mones DOC, B, S and F were simultaneously determined
in the cell culture supernatant using an UPLCMS/MS
method as described previously (25, 26, 27). Western blot
analysis was carried out to ensure the expression and
translation of WT and mutant proteins as described
previously (24, 25, 28).
All assays were performed in at least three indepen-
dent triplicate experiments, and data are presented as
meanGS.D. Kinetic parameters were established by non-
linear regression using the MichaelisMenten equation to
determine the MichaelisMenten constant (Km) and
maximum velocity (Vmax). Catalytic efficiency was defined
as the ratio Vmax/Km expressed as percentage of WT.
The 11-hydroxylase activity of the mutants was expressed
as a percentage of substrate conversion in picomole per
milligram of total protein per minute, defining CYP11B1
WT activity as 100% after correction for total protein
with Renilla luciferase activity. Enzyme kinetic para-
meters and enzymatic activity were calculated using the
GraphPad Prism Software, version 5.0 (GraphPad, Inc.,
San Diego, CA, USA).
Molecular modelling
We used a fold recognition algorithm to show that the
human CYP11B1 sequence is compatible with the archi-
tecture of the enzyme family (ProHit package, ProCeryon
Biosciences GmbH, Salzburg, Austria). The template
structure with the highest score of the pair potential
was the X-ray structure of the cytochrome CYP11A1 (PDB
accession code: 3MZS) and served as the template for the
three-dimensional model of CYP11B1. According to the
alignment obtained by the fold recognition procedure,
amino acid residues were exchanged in the template.
Insertions and deletions in CYP11B1 were modelled
using a database search approach included in the soft-
ware package WHATIF. The structural representation
was generated with the Ribbons Software (Avatar Software
AB, Stockholm, Sweden) as described previously (29).
 Clinical Study S Polat and others
Results
Sequence analysis of the CYP11B1 gene
Patient 1 showed a novel homozygous guanine deletion at
cDNA position 372 (c.372delG) in exon 2 of the CYP11B1
gene, resulting in a frameshift and a premature stop
codon (p.His125Thrfs*8). Patient 2 showed the identical
homozygous deletion c.372delG. Parents were not avail-
able for analysis; therefore, a heterogeneous deletion
of the CYP11B1 gene cannot be ruled out. Genetic analysis
in patient 3 depicted a novel homozygous duplication
of six bases in exon 8, c.1387_1392dupCTGCTG, causing
a duplication of two leucines in frame p.Leu463_
Leu464dup. The CYP11B1 gene analysis revealed that
patient 4 was compound heterozygous for the novel
mutation c.372delG (p.His125Thrfs*8) inherited from
the mother and the novel mutation c.449COT
(p.Ser150Leu) located in exon 3 inherited from the
father (Fig. 2).
European Journal of Endocrinology
In vitro functional 11-hydroxylase assays
The three novel CYP11B1 mutations were functionally
analysed using transiently transfected HEK293 cells
measuring the conversion of DOC to B and S to F. The
two mutations detected in patients with 11OHD
(p.His125Thrfs*8 and p.Leu463_Leu464dup) had no
residual enzymatic activity. The mutation p.Ser150Leu
detected with non-classic 11OHD showed only partial
11-hydroxylase impairment with a reduced activity of
19.2G1.4% (meanGS.D.) and 14.7G0.5% (meanGS.D.) for
the conversion to B and F compared with WT respectively.
Determination of kinetic constants showed similar Km
values for both B and F production with significantly
impaired Vmax compared with the WT (Fig. 3).
Western blot analysis demonstrated that all mutations
apart from the p.His125Thrfs*8 had translation efficiency
similar to WT. The mutant p.His125Thrfs*8 was not
detectable by western blotting either due to the loss of
the antibody binding site or due to nonsense-mediated
mRNA or protein decay (Supplementary Fig. 1, see section
on supplementary data given at the end of this article).
Molecular modelling
The frameshift mutation p.His125Thrfs*8 terminates the
translation at the BC loop of the protein (30). By this, all
relevant enzyme structures including the substrate recog-
nitions sides and haeme-binding sites are not present,
Three novel CYP11B1 mutations 170:5 701
resulting in a complete loss of function (31, 32). The
variation p.Leu463_Leu464dup is located in the amino
terminal L-helix. Two leucines are inserted in a
leucine-rich part of the L-helix carrying four leucines.
The L-helix is involved in haeme binding as well as in
interactions with redox partners (30). The distal L-helix
forms parts of the proteins surface. The residue p.Ser150 is
located in the CD loop connecting the C-helix with the
D-helix (Fig. 4).
Discussion
In the present study, we have functionally characterised
three novel CYP11B1 mutations detected in three
families. The p.Leu463_Leu464dup and p.Ser150Leu
mutations were unique for the studied families. On the
contrary, the p.His125Thrfs*8 mutation was detected
in both Moroccan and Turkish families where there is
no known relationship. Reported CYP11B1 mutations
are p.Arg448Cys (33, 34), p.Trp116Cys, p.Leu299Pro (2),
p.Leu489Ser (15), Glu338term (35) and p.Asn394
Argfs*74, p.Trp318Thr, p.Arg43Gln and Ala259Asp in the
Turkish population IVS5C2TOG, p.Gly446Val (36) and
p.Arg448Cys (22) in the Moroccan population. The
elevated frequency of 11OHD in Morocco and Turkey is
most likely due to the higher rate of consanguineous
marriages producing an enrichment of recessively inher-
ited diseases (37). The prevalence of consanguinity in
Morocco reported to date varies between 2028 and
59.1% in patients suffering from autosomal recessive
diseases (38). In Turkey, around 20% of marriages are
consanguineous (39).
In vitro 11-hydroxylase activity of !5% can be
considered severe and is most often associated with classic
11OHD (7, 24, 40). The patients 1 and 2 carrying a
completely inactivating mutation (p.His125Thrfs*8) of
the CYP11B1 gene show a typical classical 11OHD
phenotype with highly elevated steroids hormones,
prenatal virilisation of the external genitalia in females
and macrogenitosomia in males (5). Neither patients
showed obvious signs of hypertension. The latest clinical
assessment revealed that hydrocortisone treatment with
doses above the recommended has not been effective
enough to normalise steroid hormone levels in either
patient, most likely because of poor adherence to the
treatment. This demonstrates the necessity for long-term
follow-up of the patients by experienced physicians,
which is obviously difficult in rural Turkey. The mutation
p.His125Thrfs has a comparable effect on the protein
structure and function with the mutant c.358-362dup
www.eje-online.org
 Clinical Study S Polat and others Three novel CYP11B1 mutations 170:5 702

A c.372deIG c.1387_1392dupCTGCTG
p.His125Thrfs*8 p.Leu463_Leu464dup

1 2 3 4 5 6 7 8 9
c.449C>T
p.Ser150Leu

B
Patients 1 and 2

? ?

? ?

c.372de1G/ c.372de1G/
c.372deIG c.372deIG

Patient 3
European Journal of Endocrinology

? ?

** ** **

? ? ?

c.1387_1392dup/
c.1387_1392dup

? ?

Patient 4

c.449C>T c.372deIG
* *

c.372delG/ c.372delG/
c.449C>T c.449C>T

Figure 2
Molecular genetic analysis of the CYP11B1 gene. (A) Schematic localisation of the mutations. (B) Pedigrees of the patients from three
unrelated families with electropherograms of the mutations. Star indicates mutated nucleotides. Question marks indicate
individuals not available for genetic analysis.

(p.H122Dfs*13) detected in a patient with a classic 11OHD
phenotype described previously by Skinner et al. (41).
Patient 3, carrying a complete loss-of-function
mutation (p.Leu463_Leu464dup) on the CYP11B1 gene,
came to clinical attention with premature pubarche in
childhood. He was short statured due to insufficient
treatment during childhood and adolescence. He
developed bilateral TART which is the most important
cause of infertility in men with CAH to 21-hydroxylase
deficiency (42). Ectopically located adrenal tissues

www.eje-online.org
 Clinical Study S Polat and others Three novel CYP11B1 mutations 170:5 703

A C
100 100

percentage of WT activity

percentage of WT activity
DOC to B conversion in
80 80

S to F conversion in
60 60

40 40

20 20

0 0
WT Ser150Leu pcDNA WT Ser150Leu pcDNA

B D
0.6 0.6
WT WT
0.5 pSer150Leu 0.5 pSer150Leu
1/v (pmol/min1 per mg1)

1/v (pmol/min1 per mg1)

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0
2 0 2 4 6 8 10 2 0 2 4 6 8 10
1/DOC (M1) 1/S (M1)
European Journal of Endocrinology

DOC B WT p.Ser150Leu S F WT p.Ser150Leu

Vmax 20012 344 Vmax 1066 172
Km 1.20.8 1.00.3 Km 0.60.1 0.70.8
Vmax/Km 167 34 Vmax/Km 176 24
Catalytic 100 20 Catalytic 100 14
efficiency (%) efficiency (%)

Figure 3
Residual 11-hydroxylase activity of the CYP11B1 variants corticosterone. One data point represents the mean of a
assessed in transiently transfected HEK293 cells co-expressing triplicate detection. (C) Cortisol production of the mutant
human WT or mutant CYP11B1 with human Adx reductase and expressed as percentage of WT activity which is defined as
Adx respectively. (A) Corticosterone production of the mutant 100%. Error bars represent the meanGS.D. (D) LineweaverBurk
expressed as percentage of WT activity which is defined as plot of 11-hydroxylase activity converting 11-deoxycortisol
100%. Error bars represent the meanGS.D. (B) LineweaverBurk to cortisol. One data point represents the mean of a
plot of 11-hydroxylase activity converting DOC to triplicate detection.

usually regress with advancing age except in CAH 21-hydroxylase deficiency (49, 50). Therefore, the findings
patients in whom low levels of cortisol induce ACTH in 11OHD may be a collection bias. In the patient, the
secretion and hyperplasia of dispersed adrenal tissue (43). TART located in the right testis was removed because of
The presence of adrenal rests within the testes of adult its size and the difficulty to distinguish it from a Leydig
males with classic 21-hydroxylase CAH is more frequent in cell tumour during follow-up (51). TART cells showed
the salt-wasting form and is associated with a higher risk eosinophilic cytoplasm with neither lipochrome pigments
for infertility (44). 11OHD patients with TART are rare. nor Reinke crystalloids (Fig. 1) (47, 52). The tumour on
Up to our knowledge, only nine cases are presented in the the left is under control with prednisolone treatment.
literature so far (43, 45, 46, 47, 48). This is most likely due Whether surgery has had a positive effect on the
to the smaller incidence of CAH due to 11-hydroxylase testicular function or whether resuming prednisolone
deficiency. All reported cases were poorly compliant to treatment led to fertility remains unclear. TART surgery
treatment. However, the idea that poor control may lead in 21-hydroxylase deficiency has no proven effect on
to a higher risk of TART was just recently refuted in fertility (53).

www.eje-online.org
 Clinical Study S Polat and others
S150
Figure 4
European Journal of Endocrinology
Ribbon representation of the three-dimensional molecular
model of CYP11B1. Helix C, the CD-loop and helix D are
coloured light blue, red and light green respectively. The side
chain of residue Ser150 located in the CD loop is depicted.
The variation p.Leu463_Leu464dup detected in
patient 3 is located in the amino terminal L-helix. Two
leucines are inserted in a leucine-rich part of the L-helix
carrying four leucines. The L-helix is involved in haeme
binding as well as in interactions with redox partners (30).
The distal L-helix forms parts of the proteins surface.
As four residues are needed for a full helical turn, the
addition of two residues will produce an additional half
turn of the helix, leading to a displacement of the pro-
ximal part of the helix and disturbance of substrate
binding and electron flux, or interference with the binding
of redox partners due to changes in the protein surface.
Both possibilities will result in a complete loss of function.
Interestingly, Geley et al. described a patient with classic
11OHD, caused by a duplication of a single leucine at
the identical position. This variant also caused a complete
loss of 11-hydroxylase activity (7).
Patient 4 showed clinical signs compatible with non-
classic CAH (25). The novel mutation p.Ser150Leu
detected in this patient had a residual activity of 19.2%
of the WT. By this, it is comparable with the previously
described mutations associated with non-classic 11OHD.
Interestingly, only 12 mutations causing non-classic
11OHD have been described so far (8, 10, 25, 54). This is
most likely due to the overall rarity of 11OHD. However,
www.eje-online.org
Three novel CYP11B1 mutations 170:5 704
the increasing number of reports on affected patients
should increase the awareness for non-classic 11OHD. The
residual activity of mutations associated with non-classic
11OHD ranged from 9 to 40% of WT activity, which is
well comparable with the residual activity of mutations in
the CYP21A2 gene causing non-classic 21-hydroxylase
deficiency (55). The residue p.Ser150 is located in the
CD loop connecting the C-helix with the D-helix of
the CYP11B1 protein (Fig. 4). This loop is located at the
surface of the molecule. The change to p.Leu150 does
not lead to obvious steric problems with neighbouring
residues in silico. However, the exchange of the polar
serine by the hydrophobic leucine leads to a change in the
surface properties of the molecule. Since the adjacent
D-helix is part of the redox partner interaction side, the
mutation may induce a decrease in electron flux which is
consistent with the observed reduction of Vmax (30, 32).
In conclusion, we have shown the inactivating nature
of two novel CYP11B1 mutations in classic 11OHD in
addition to one variant with residual activity associated
with non-classic 11OHD. The analyses of in vitro enzyme
function broaden our understanding of CYP11B1 function
relationships and help to counsel families as the severity
of the clinical disease expression can be estimated. The
condition of non-classic 11OHD is a rare disease but
should not be missed in the differential diagnosis of
hyperandrogenism.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/
EJE-13-0737.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This study was partially supported by an unrestricted visiting scholarship
grant from the European Society for Paediatric Endocrinology to S Polat.
Acknowledgements
The authors are grateful to Rita Bernhardt (Institute of Biochemistry,
Universitat des Saarlandes, Saarbrucken, Germany) for providing the
CYP11B1 cDNA, Walter L Miller (Department of Pediatrics, University of
California, San Francisco, CA, USA) for providing the Adx and Adr cDNA and
Hiroshi Takemori (Department of Molecular Physiological Chemistry, Osaka
University Medical School, Osaka, Japan) for providing the antihuman-
CYP11B rabbit antiserum. We appreciate the expert technical assistance
of Tanja Stampe and Gisela Hohmann.
 Clinical Study S Polat and others
References
1 White PC & Speiser PW. Steroid 11b-hydroxylase deficiency and related
disorders. Endocrinology and Metabolism Clinics of North America 1994 23
325339.
2 Krone N, Riepe FG, Gotze D, Korsch E, Rister M, Commentz J,
Partsch CJ, Grotzinger J, Peter M & Sippell WG. Congenital adrenal
hyperplasia due to 11-hydroxylase deficiency: functional character-
ization of two novel point mutations and a three-base pair deletion in
the CYP11B1 gene. Journal of Clinical Endocrinology and Metabolism 2005
90 37243730. (doi:10.1210/jc.2005-0089)
3 White PC, Curnow KM & Pascoe L. Disorders of steroid 11b-hydroxylase
isozymes. Endocrine Reviews 1994 15 421438.
4 New MI & White PC. Genetic disorders of steroid hormone synthesis
and metabolism. Baillie`res Clinical Endocrinology and Metabolism 1995 9
525554. (doi:10.1016/S0950-351X(95)80587-7)
5 Zachmann M, Tassinari D & Prader A. Clinical and biochemical
variability of congenital adrenal hyperplasia due to 11b-hydroxylase
deficiency. A study of 25 patients. Journal of Clinical Endocrinology and
Metabolism 1983 56 222229. (doi:10.1210/jcem-56-2-222)
6 Stenson PD, Ball E, Howells K, Phillips A, Mort M & Cooper DN. Human
Gene Mutation Database: towards a comprehensive central mutation
database. Journal of Medical Genetics 2008 45 124126. (doi:10.1136/
jmg.2007.055210)
7 Geley S, Kapelari K, Johrer K, Peter M, Glatzl J, Vierhapper H, Schwarz S,
European Journal of Endocrinology
Helmberg A, Sippell WG, White PC et al. CYP11B1 mutations causing
congenital adrenal hyperplasia due to 11b-hydroxylase deficiency.
Journal of Clinical Endocrinology and Metabolism 1996 81 28962901.
8 Parajes S, Loidi L, Reisch N, Dhir V, Rose IT, Hampel R, Quinkler M,
Conway GS, Castro-Feijoo L, Araujo-Vilar D et al. Functional con-
sequences of seven novel mutations in the CYP11B1 gene: four
mutations associated with nonclassic and three mutations causing
classic 11{b}-hydroxylase deficiency. Journal of Clinical Endocrinology and
Metabolism 2010 95 779788. (doi:10.1210/jc.2009-0651)
9 Chabre O, Portrat-Doyen S, Vivier J, Morel Y & Defaye G. Two novel
mutations in splice donor sites of CYP11B1 in congenital adrenal
hyperplasia due to 11b-hydroxylase deficiency. Endocrine Research 2000
26 797801. (doi:10.3109/07435800009048602)
10 Joehrer K, Geley S, Strasser-Wozak EM, Azziz R, Wollmann HA,
Schmitt K, Kofler R & White PC. CYP11B1 mutations causing non-
classic adrenal hyperplasia due to 11b-hydroxylase deficiency. Human
Molecular Genetics 1997 6 18291834. (doi:10.1093/hmg/6.11.1829)
11 Speiser PW & White PC. Congenital adrenal hyperplasia. New England
Journal of Medicine 2003 349 776788. (doi:10.1056/NEJMra021561)
12 Wu C, Zhou Q, Wan L, Ni L, Zheng C, Qian Y & Jin J. Novel
homozygous p.R454C mutation in the CYP11B1 gene leads to
11b-hydroxylase deficiency in a Chinese patient. Fertility and Sterility
2011 1122 e1123e1126.
13 Krone N, Grotzinger J, Holterhus PM, Sippell WG, Schwarz HP &
Riepe FG. Congenital adrenal hyperplasia due to 11-hydroxylase
deficiency insights from two novel CYP11B1 mutations (p.M92X,
p.R453Q). Hormone Research 2009 72 281286. (doi:10.1159/
000245930)
14 Sonino N, Levine LS, Vecsei P & New MI. Parallelism of 11b- and
18-hydroxylation demonstrated by urinary free hormones in man.
Journal of Clinical Endocrinology and Metabolism 1980 51 557560.
(doi:10.1210/jcem-51-3-557)
15 Peters CJ, Nugent T, Perry LA, Davies K, Morel Y, Drake WM, Savage MO
& Johnston LB. Cosegregation of a novel homozygous CYP11B1
mutation with the phenotype of non-classical congenital adrenal
hyperplasia in a consanguineous family. Hormone Research 2007 67
189193. (doi:10.1159/000097244)
16 Gokcay G, Furman A & Neyzi O. Updated growth curves for Turkish
children aged 15 days to 60 months. Child: Care, Health and Development
2008 34 454463. (doi:10.1111/j.1365-2214.2008.00813.x)
Three novel CYP11B1 mutations 170:5 705
17 Kondolot M, Balci E, Ozturk A, Mazicioglu MM, Hatipoglu N,
Kurtoglu S & Ustunbas HB. Body mass index percentiles for Turkish
children aged 084 months. Annals of Human Biology 2011 38 676680.
(doi:10.3109/03014460.2011.605800)
18 Tumer N, Yalcinkaya F, Ince E, Ekim M, Kose K, Cakar N, Kara N,
Ozkaya N, Ensari C & Onder S. Blood pressure nomograms for children
and adolescents in Turkey. Pediatric Nephrology 1999 13 438443.
(doi:10.1007/s004670050636)
19 Cinaz P, Yesilkaya E, Onganlar YH, Boyraz M, Bideci A, Camurdan O &
Karaoglu AB. Penile anthropometry of normal prepubertal boys in
Turkey. Acta Paediatrica 2012 101 e33e36. (doi:10.1111/j.1651-2227.
2011.02386.x)
20 Neyzi O, Furman A, Bundak R, Gunoz H, Darendeliler F & Bas F. Growth
references for Turkish children aged 6 to 18 years. Acta Paediatrica 2006
95 16351641. (doi:10.1080/08035250600652013)
21 Ozturk A, Mazicioglu MM, Hatipoglu N, Budak N, Keskin G, Yazlak Z,
Balci N, Yildiz H, Yildiz K, Ustunbas HB et al. Reference body mass index
curves for Turkish children 6 to 18 years of age. Journal of Pediatric
Endocrinology & Metabolism 2008 21 827836. (doi:10.1515/JPEM.2008.
21.9.827)
22 White PC, Dupont J, New MI, Leiberman E, Hochberg Z & Rosler A. A
mutation in CYP11B1 (Arg-448His) associated with steroid
11b-hydroxylase deficiency in Jews of Moroccan origin. Journal of
Clinical Investigation 1991 87 16641667. (doi:10.1172/JCI115182)
23 Naiki Y, Kawamoto T, Mitsuuchi Y, Miyahara K, Toda K, Orii T, Imura H
& Shizuta Y. A nonsense mutation (TGG [Trp116]/TAG [Stop]) in
CYP11B1 causes steroid 11b-hydroxylase deficiency. Journal of Clinical
Endocrinology and Metabolism 1993 77 16771682.
24 Krone N, Grischuk Y, Muller M, Volk RE, Grotzinger J, Holterhus PM,
Sippell WG & Riepe FG. Analyzing the functional and structural
consequences of two point mutations (P94L and A368D) in the
CYP11B1 gene causing congenital adrenal hyperplasia resulting from
11-hydroxylase deficiency. Journal of Clinical Endocrinology and Metab-
olism 2006 91 26822688. (doi:10.1210/jc.2006-0209)
25 Menabo S, Polat S, Baldazzi L, Kulle AE, Holterhus PM, Grotzinger J,
Fanelli F, Balsamo A & Riepe FG. Congenital adrenal hyperplasia due to
11-b-hydroxylase deficiency: functional consequences of four CYP11B1
mutations. European Journal of Human Genetics 2013. (doi:10.1038/
ejhg.2013.197)
26 Kulle AE, Riepe FG, Melchior D, Hiort O & Holterhus PM. A novel
ultrapressure liquid chromatography tandem mass spectrometry
method for the simultaneous determination of androstenedione,
testosterone, and dihydrotestosterone in pediatric blood samples:
age- and sex-specific reference data. Journal of Clinical Endocrinology and
Metabolism 2010 95 23992409. (doi:10.1210/jc.2009-1670)
27 Kulle AE, Welzel M, Holterhus PM & Riepe FG. Principles and clinical
applications of liquid chromatography tandem mass spectrometry for
the determination of adrenal and gonadal steroid hormones. Journal of
Endocrinological Investigation 2011 34 702708.
28 Clark BJ, Wells J, King SR & Stocco DM. The purification, cloning, and
expression of a novel luteinizing hormone-induced mitochondrial
protein in MA-10 mouse Leydig tumor cells. Characterization of the
steroidogenic acute regulatory protein (StAR). Journal of Biological
Chemistry 1994 269 2831428322.
29 Kraulis PJ. Molscript a program to produce both detailed and
schematic plots of protein structures. Journal of Applied Crystallography
1991 24 946950. (doi:10.1107/S0021889891004399)
30 Otyepka M, Skopalik J, Anzenbacherova E & Anzenbacher P. What
common structural features and variations of mammalian P450s are
known to date? Biochimica et Biophysica Acta 2007 1770 376389.
(doi:10.1016/j.bbagen.2006.09.013)
31 Gotoh O. Substrate recognition sites in cytochrome-P450 family-2
(Cyp2) proteins inferred from comparative analyses of amino-acid and
coding nucleotide-sequences. Journal of Biological Chemistry 1992 267
8390.
www.eje-online.org
 Clinical Study S Polat and others
32 Robins T, Carlsson J, Sunnerhagen M, Wedell A & Persson B. Molecular
model of human CYP21 based on mammalian CYP2C5: structural
features correlate with clinical severity of mutations causing congenital
adrenal hyperplasia. Molecular Endocrinology 2006 20 29462964.
(doi:10.1210/me.2006-0172)
33 Cingoz S, Ozkan B, Doneray H & Sakizli M. Familial pericentric
inversion chromosome 3 and R448C mutation of CYP11B1 gene in
Turkish kindred with 11b-hydroxylase deficiency. Journal of Endocrino-
logical Investigation 2007 30 285291.
34 Helmberg A, Ausserer B & Kofler R. Frame shift by insertion of 2
basepairs in codon 394 of CYP11B1 causes congenital adrenal
hyperplasia due to steroid 11b-hydroxylase deficiency. Journal of
Clinical Endocrinology and Metabolism 1992 75 12781281.
35 Zhu YS, Cordero JJ, Can S, Cai LQ, You XK, Herrera C, DeFillo-Ricart M,
Shackleton C & Imperato-McGinley J. Mutations in CYP11B1 gene:
phenotypegenotype correlations. American Journal of Medical Genetics.
Part A 2003 122A 193200. (doi:10.1002/ajmg.a.20108)
36 Chabraoui L, Abid F, Menassa R, Gaouzi A, El Hessni A & Morel Y. Three
novel CYP11B1 mutations in congenital adrenal hyperplasia due to
steroid 11b-hydroxylase deficiency in a Moroccan population. Hormone
Research in Paediatrics 2010 74 182189. (doi:10.1159/000281417)
37 Hussain R, Bittles AH & Sullivan S. Consanguinity and early mortality
in the Muslim populations of India and Pakistan. American Journal of
Human Biology 2001 13 777787. (doi:10.1002/ajhb.1124)
38 Jaouad IC, Elalaoui SC, Sbiti A, Elkerh F, Belmahi L & Sefiani A.
Consanguineous marriages in Morocco and the consequence for the
European Journal of Endocrinology
incidence of autosomal recessive disorders. Journal of Biosocial Science
2009 41 575581. (doi:10.1017/S0021932009003393)
39 Alper OM, Erengin H, Manguoglu AE, Bilgen T, Cetin Z, Dedeoglu N &
Luleci G. Consanguineous marriages in the province of Antalya,
Turkey. Annales de Genetique 2004 47 129138.
40 Kuribayashi I, Nomoto S, Massa G, Oostdijk W, Wit JM,
Wolffenbuttel BHR, Shizuta Y & Honke K. Steroid 11-b-hydroxylase
deficiency caused by compound heterozygosity for a novel mutation,
p.G314R, in one CYP11B1 allele, and a chimeric CYP11B2/CYP11B1
in the other allele. Hormone Research 2005 63 284293. (doi:10.1159/
000087074)
41 Skinner CA & Rumsby G. Steroid 11b-hydroxylase deficiency caused
by a five base pair duplication in the CYP11B1 gene. Human Molecular
Genetics 1994 3 377378. (doi:10.1093/hmg/3.2.377)
42 Claahsen-van der Grinten HL, Hermus AR & Otten BJ. Testicular
adrenal rest tumours in congenital adrenal hyperplasia. International
Journal of Pediatric Endocrinology 2009 2009 624823. (doi:10.1186/
1687-9856-2009-624823)
43 Karnak I, Senocak ME, Gogus S, Buyukpamukcu N & Hicsonmez A.
Testicular enlargement in patients with 11-hydroxylase deficiency.
Journal of Pediatric Surgery 1997 32 756758. (doi:10.1016/S0022-
3468(97)90027-0)
44 Cabrera MS, Vogiatzi MG & New MI. Long term outcome in adult males
with classic congenital adrenal hyperplasia. Journal of Clinical
Endocrinology and Metabolism 2001 86 30703078.
www.eje-online.org
Three novel CYP11B1 mutations 170:5 706
45 Srikanth MS, West BR, Ishitani M, Isaacs H Jr, Applebaum H & Costin G.
Benign testicular tumors in children with congenital adrenal hyper-
plasia. Journal of Pediatric Surgery 1992 27 639641. (doi:10.1016/0022-
3468(92)90466-K)
46 Ghazi AA, Hadayegh F, Khakpour G, Azizi F & Melby JC. Bilateral
testicular enlargement due to adrenal remnant in a patient with C11
hydroxylase deficiency congenital adrenal hyperplasia. Journal of
Endocrinological Investigation 2003 26 8487.
47 Mirzaei MR, Rezvanian H, Siavash M, Parham M & Mahzouni P.
A patient with refractory testicular adrenal rest tumour in the setting
of cyp11b1 deficiency congenital adrenal hyperplasia. BMJ Case
Reports 2009. (doi:10.1136/bcr.06.2008.0280)
48 Charfi N, Kamoun M, Feki Mnif M, Mseddi N, Mnif F, Kallel N,
Ben Naceur B, Rekik N, Fourati H, Daoud E et al. Leydig cell tumor
associated with testicular adrenal rest tumors in a patient with
congenital adrenal hyperplasia due to 11b-hydroxylase deficiency. Case
Reports in Urology 2012 2012 648643. (doi:10.1155/2012/648643)
49 Bercovici JP, Fiet J, Gibault L, Volant A, Abalain JH, Floch HH, Sonnet E
& Fournier G. Testicular adrenal rest tumours in salt wasting congenital
adrenal hyperplasia (in vivo and in vitro studies). Journal of Steroid
Biochemistry and Molecular Biology 2005 93 6772. (doi:10.1016/
j.jsbmb.2004.10.023)
50 Reisch N, Rottenkolber M, Greifenstein A, Krone N, Schmidt H,
Reincke M, Schwarz HP & Beuschlein F. Testicular adrenal rest tumors
develop independently of long-term disease control: a longitudinal
analysis of 50 adult men with congenital adrenal hyperplasia due to
classic 21-hydroxylase deficiency. Journal of Clinical Endocrinology and
Metabolism 2013 98 E1820E1826. (doi:10.1210/jc.2012-3181)
51 Rich MA & Keating MA. Leydig cell tumors and tumors associated with
congenital adrenal hyperplasia. Urologic Clinics of North America 2000
27 519528 x. (doi:10.1016/S0094-0143(05)70099-9)
52 Mostofi FK, Davis J Jr & Rehm S. Tumours of the testis. IARC Scientific
Publications 1994 111 407429.
53 Claahsen-van der Grinten HL, Otten BJ, Takahashi S, Meuleman EJ,
Hulsbergen-van de Kaa C, Sweep FC & Hermus AR. Testicular adrenal
rest tumors in adult males with congenital adrenal hyperplasia:
evaluation of pituitarygonadal function before and after successful
testis-sparing surgery in eight patients. Journal of Clinical Endocrinology
and Metabolism 2007 92 612615. (doi:10.1210/jc.2006-1311)
54 Reisch N, Hogler W, Parajes S, Rose IT, Dhir V, Gotzinger J, Arlt W &
Krone N. A diagnosis not to be missed: non-classic steroid
11b-hydroxylase deficiency presenting with premature adrenarche and
hirsutism. Journal of Clinical Endocrinology and Metabolism 2013 98
E1620E1625. (doi:10.1210/jc.2013-1306)
55 White PC & Speiser PW. Congenital adrenal hyperplasia due to
21-hydroxylase deficiency. Endocrine Reviews 2000 21 245291.
56 Kulle AE, Welzel M, Holterhus PM & Riepe FG. Implementation of a
liquid chromatography tandem mass spectrometry assay for eight
adrenal C-21 steroids and pediatric reference data. Hormone Research in
Paediatrics 2013 79 2231. (doi:10.1159/000346406)
Received 8 September 2013
Revised version received 15 December 2013
Accepted 14 February 2014